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PubMed2004

Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans.

UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer.

Amino Acid Sequence↗
PubMed2005

The Nramp orthologue of Cryptococcus neoformans is a pH-dependent transporter of manganese, iron, cobalt and nickel.

Cryptococcus neoformans is an important human opportunistic pathogen and a facultative intracellular parasite, particularly in HIV-infected individuals. Little is known about metal ion transport in this organism. C. neoformans encodes a single member of the Nramp (natural resistance-associated macrophage protein) family of bivalent cation transporters, known as Cramp, which we have cloned and expressed in Xenopus laevis oocytes and Spodoptera frugiperda Sf 21 insect cells. Cramp induces saturable transport of a broad range of bivalent transition series cations, including Mn2+, Fe2+, Co2+ and Ni2+. Maximal cation transport occurs at pH 5.5-6.0, consistent with the proton gradient-based energetics of other Nramp orthologues. Mn2+ transport is diminished in the presence of 140 mM Na+, compatible with a Na+ slippage mechanism proposed for the Saccharomyces cerevisiae Nramp orthologue Smf1p. Cramp resembles Smf1p with respect to predicted membrane topology, substrate specificity and pH dependence, but differs in terms of its apparent affinity for Mn2+ and negligible inhibition by Zn2+. Cramp is the first Nramp orthologue from a fungal pathogen to be functionally characterized. Insights afforded by these findings will allow the formulation of new hypotheses regarding the role of metal ions in the pathophysiology of cryptococcosis.

Animals↗
PubMed1996

Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing.

A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an alpha-cyclodextrin-Sepharose 6B column, and was characterized as an alpha-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified alpha-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-starch-digesting and raw-starch-absorbing activities. Furthermore it was shown to be thermostable. An open reading frame of the cDNA specified 611 amino acids, including a putative signal peptide of 20 amino acids. The N-terminal region of AMY-CS2 (from the N-terminus to position 496) had 49.7% similarity with the whole region of alpha-amylase from Aspergillus oryzae (Taka-amylase), whereas the C-terminal region had a sequence that was similar to the C-terminal region of glucoamylase G1 from A. niger. In addition, putative raw-starch-binding motifs exist in some amylolytic enzymes. A mutant AMY-CS2 that lacks the C-terminal domain lost not only its ability to bind or digest raw starch, but also its thermostability. Consequently it is possible that the putative raw-starch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability.

Amino Acid Sequence↗
PubMed1996

IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans.

We examined the role of IL-12 in host resistance to Cryptococcus neoformans using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with viable yeast cells. Mice untreated with IL-12 allowed an uncontrolled multiplication of yeast cells in the lung with infiltrations of few inflammatory cells, and a cryptococcal dissemination to the brain and meningitis by 3 weeks, resulting in death of all animals within 4-6 weeks. IL-12, when administered from the day of tracheal infection for 7 days, induced a marked infiltration of inflammatory cells, consisting mostly of mononuclear cells, and significantly reduced the number of viable yeast cells in the lung. The treatment suppressed brain dissemination, as shown by a marked reduction of yeast cells in the brain and prevention of meningitis. These effects resulted in a significant increase in the survival rate of infected mice. In contrast, late administration of IL-12 commencing on day 7 after instillation of yeast cells failed to protect the mice against infection with C. neoformans. In further experiments, early administration of IL-12 markedly induced interferon-gamma (IFN-gamma) mRNA in the lungs of infected mice, while no IFN-gamma mRNA was detected without this treatment. Our results indicate that IL-12 is effective when administered in the early period of pulmonary cryptococcal infection.

Animals↗
PubMed1996

Generation of biologically active anti-Cryptococcus neoformans IgG, IgE and IgA isotype switch variant antibodies by acridine orange mutagenesis.

Administration of MoAbs to Cryptococcus neoformans capsular glucuronoxylomannan (GXM) can alter the course of infection in mouse models. However, the effectiveness of these antibodies appears to depend on isotype and specificity. Comparison of isotype protection efficacy requires families of MoAbs with identical fine specificity and different constant region domain. The generation of such families by hybridoma technology is not always possible because the immune response produces MoAbs of limited classes or subclasses. In these instances isotype switch variants can be isolated in vitro. Unfortunately, standard methods of recovering spontaneous switch variants are often unsuccessful, mainly because of the low frequency of switching. In this study we demonstrate that acridine orange stimulation of an IgG3 anti-C. neoformans-producing hybridoma can be used to recover the entire set of isotype switch variants: IgGl, IgG2b, IgG2a, IgE and IgA. All isotype switch variants bind to GXM; fine specificity mapping, using an 11 amino acid peptide polysaccharide mimetope, revealed conservation of binding site specificity. Furthermore, all isotype switch variants reacted with an anti-idiotopic MoAb. The functional activity of this set of MoAbs was demonstrated by their ability to enhance phagocytosis and antifungal efficacy of human macrophage-like THP-1 cells, with IgG3 being the most effective and IgE being the least effective.

Acridine Orange↗
PubMed1999

Interferon-gamma (IFN-gamma)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected with Cryptococcus neoformans.

We have recently demonstrated that IL-12 induced cellular inflammatory responses consisting mainly of accumulation of mononuclear leucocytes in the lungs of mice infected with Cryptococcus neoformans and protected mice against fulminant infection. We examined the involvement of endogenously synthesized IFN-gamma in such a response by investigating the effects of a neutralizing monoclonal antibody against this cytokine. The latter treatment completely abrogated the positive effects of IL-12 on survival of infected mice and prevented IL-12-induced elimination of microbials from the lungs. Histopathological examination showed that accumulation of mononuclear leucocytes in the infected lungs caused by IL-12 was clearly inhibited by anti-IFN-gamma MoAb. We also examined the local production of mononuclear cell-attracting chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and IFN-gamma-inducible protein 10 (IP-10) in the lungs using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that these chemokines were not synthesized in the infected lungs, while IL-12 treatment markedly induced their production. Interestingly, neutralizing anti-IFN-gamma MoAb strongly suppressed IL-12-induced production of these chemokines. Similar results were obtained with MCP-1 and MIP-1alpha when their synthesis was measured at the protein level using respective ELISA kits. Our results indicate that IFN-gamma plays a central role in the protective effects of IL-12 by inducing mononuclear leucocyte-attracting chemokines and cellular inflammatory responses.

Animals↗
PubMed1999

Pulmonary surfactant protein A binds to Cryptococcus neoformans without promoting phagocytosis.

BACKGROUND: Evidence is accumulating that the alveolar collecting surfactant protein A (SP-A) plays an important role in the first line of defence against infiltrating pathogenic micro-organisms and viruses. The ability of SP-A to facilitate the binding and uptake of acapsular Cryptococcus neoformans by monocyte-derived macrophages, human alveolar macrophages, monocytes and polymorphonuclear leucocytes was investigated. MATERIALS AND METHODS: Binding, competition and phagocytosis experiments were performed using a flow cytometry technique. RESULTS: SP-A bound to both the acapsular and the encapsulated form of C. neoformans in a concentration-dependent manner. SP-A showed a threefold better binding to the acapsular yeast: this binding was partly calcium dependent and could be inhibited by mannose (ID50 = 3 mmol L-1) and glucose (ID50 = 2.1 mmol L-1) but not by galactose (ID50 = 391 mmol L-1). SP-A did not function as an opsonin in phagocytosis of acapsular C. neoformans for any of the phagocytes studied. CONCLUSION: Our results indicate that SP-A binds in a concentration-dependent manner to both encapsulated and acapsular C. neoformans. Despite SP-A binding to the acapsular C. neoformans, phagocytosis by various phagocytes was not enhanced.

Animals↗
PubMed1997

CR3-dependent phagocytosis by murine macrophages: different cytokines regulate ingestion of a defined CR3 ligand and complement-opsonized Cryptococcus neoformans.

Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti-phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non-immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1-2 organisms per cell). Previously we have shown that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) efficiently enhance ingestion of serum-opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6-8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement-opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3-dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.

Animals↗
PubMed1997

Both CD4+ and CD8+ human lymphocytes are activated and proliferate in response to Cryptococcus neoformans.

The current studies were performed to determine the contribution of T-cell subsets to lymphocyte proliferation in response to Cryptococcus neoformans, the most common invasive mycosis in acquired immune deficiency syndrome. We demonstrate for the first time that both human CD4 and CD8 cells are activated in response to C. neoformans. Both CD4 and CD8 cells express interleukin-2 receptor alpha (IL-2R alpha) and transferrin receptor and proliferate in response to C, neoformans, however proliferation of CD8 cells was dependent upon CD4 cells. The requirement for CD4 cells was complex, since CD8 enriched cells failed to express mRNA for IL-2, suggesting that CD4-dependent IL-2 production was required for CD8-cell proliferation. However, IL-2 was not sufficient to restore CD8-cell proliferation. These studies provide experimental evidence in humans to support the clinical impression that CD4 cells are important in cryptococcosis, and suggest that the appropriate CD4-derived signals could allow CD8 cells to assist in host defence.

Adult↗
PubMed1999

B7 costimulatory ligand regulates development of the T-cell response to Cryptococcus neoformans.

The contribution of B7 molecules to the induction and maintenance of the T-cell response to the human pathogenic fungus Cryptococcus neoformans was investigated. T-cell activation by C. neoformans was regulated by B7 molecules. This costimulatory signal was necessary for initiation and maintenance of the T-cell response, through early and late requirements for B7-CD28 interaction. Blocking B7-2 inhibited the normal T-cell proliferative response. This inhibition was due, in part, to a reduced capability of T cells to produce interleukin-2 (IL-2). In contrast, the same T-cell population produced more interferon-gamma. Suppression of the normal lymphoproliferation and IL-2 secretion responses to encapsulated C. neoformans by antibodies to B7 was largely reversed by addition of the monoclonal antibody 2H1, that is reactive with the major capsular polysaccharide, glucuronoxylomannan. Overall, our data indicate that B7 molecules play a critical role in T-cell activation by C. neoformans and suggest that appropriate manipulation could drive T helper type 1 cell development.

Antibodies, Monoclonal↗
PubMed2002

Antibody to Cryptococcus neoformans capsular glucuronoxylomannan promotes expression of interleukin-12Rbeta2 subunit on human T cells in vitro through effects mediated by antigen-presenting cells.

The results reported herein show that T cells responding to encapsulated Cryptococcus neoformans cells had reduced expression of interleukin-12 receptor beta2 (IL-12Rbeta2) in comparison to those responding to non-encapsulated cells. This suggested that encapsulation with glucuronoxylomannan (GXM), the principal constituent of the C. neoformans polysaccharide antiphagocytic capsule, inhibited expression of the IL-12Rbeta2 subunit on T cells responding to cryptococcal antigens. Addition of GXM-binding monoclonal antibody (mAb) overcame this effect by promoting IL-12Rbeta2 expression and by decreasing IL-1R expression on T cells. This effect may be a consequence of mAb-induced changes on antigen-presenting cells (APC) that are closely related to increased phagocytosis. Blocking of phagocytosis with monoiodacetic acid (MIA) precluded up-regulation of B7 expression on APC and was associated with diminished IL-12Rbeta2 expression on T cells. The observed effects on T cells were interpreted as a consequence of increased APC function due to enhanced phagocytosis. These findings suggest a mechanism by which specific antibody can promote the polarization of the cellular immune response towards a Th1-like response and thus contribute to an enhanced cellular immune response against C. neoformans.

Antibodies, Fungal↗
PubMed2000

Treatment of experimental Cryptococcus neoformans infection in newborn rabbits by airway instillation of specific antibody and surfactant.

Cryptococcosis in AIDS patients has a slow response to antifungal chemotherapy, and passive antibody treatment has thus been considered as an adjunct. Polyclonal anticryptococcal IgG dissolved in a suspension of modified natural surfactant was given intratracheally to near-term rabbits. Killing of Cryptococcus neoformans within the lungs was determined by counting the colony forming units (cfu). After 5 h a significant decrease in cfu was observed in rabbits treated with the IgG-surfactant mixture compared with control animals receiving saline. In conjunction with conventional therapy, the combined treatment of IgG-surfactant given by bronchoscopy might be used in high-risk patients to enhance killing of the yeast within the lungs.

Animals↗
PubMed1998

Simultaneous infection with Blastomyces dermatitidis and Cryptococcus neoformans.

In this report, we present two cases of simultaneous infection with Blastomyces dermatitidis and Cryptococcus neoformans. In both cases, fungi were isolated from the lungs. The clinical manifestations were not suggestive of infection with two fungi. One patient was receiving immunosuppressive therapy and another was otherwise immunocompetent. A review of the literature failed to uncover any reports of co-infection with these two fungal pathogens. We discuss possible mechanisms for the isolation of both pathogens.

Aged↗
PubMed1999

Antifungal activity of cerebrospinal fluid against Cryptococcus neoformans and Candida species.

The effect of human cerebrospinal fluid (CSF) on the growth of Cryptococcus neoformans and Candida species was tested in RPMI-1640. CSF alone was highly fungistatic for both yeasts and inhibited growth in a concentration-dependent manner. Unlike human serum, CSF did not collaborate with fluconazole for killing C. neoformans. Molecular sieve fractionation of CSF on a G-200 Sephadex column yielded a highly antifungal fraction with a molecular weight around 66 kDa. On SDS-PAGE this fraction migrated as a major and a minor band corresponding to the mobility of bovine serum albumin. These novel findings suggest that CSF contains a factor(s) that provides resistance to the growth of C. neoformans or Candida species.

Animals↗
PubMed2000

Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans.

Cryptococcus neoformans (= Filobasidiella neoformans) is a significant emerging fungal pathogen of humans. To understand the evolution of this pathogen, 34 strains were obtained from various locations around the world and fragments of four genes were sequenced from each. These strains represented all three varieties and five serotypes. The four sequenced genes are: (i) the mitochondrial large ribosomal subunit RNA; (ii) the internal transcribed spacer region of the nuclear rRNA, including ITS1, 5.8S rRNA subunit and ITS2; (iii) orotidine monophosphate pyrophosphorylase; and (iv) diphenol oxidase. Phylogenetic analyses indicated considerable divergence among lineages, which corresponded to the current classification of C. neoformans into three varieties. However, there is no apparent phylogeographic pattern. Significant incongruences were observed among gene genealogies. The analyses indicated that the major lineages in C. neoformans diverged tens of millions of years ago but have undergone recent dispersion and hybridization.

Catechol Oxidase↗
PubMed1997

The Cryptococcus neoformans STE12alpha gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific.

Cryptococcus neoformans possesses two mating types, MATalpha and MATa. Alpha-Cells are more virulent than a-cells and are also, unlike a-cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFalpha, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12alpha gene, however, has one striking difference from other fungal STE12 genes; it is found only in alpha-cells. The existence of STE12alpha in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

Amino Acid Sequence↗
PubMed2000

RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans.

Cryptococcus neoformans is a basidiomycete yeast and opportunistic human pathogen of increasing clinical importance due to the increasing population of immunocompromised patients. To further investigate signal transduction cascades regulating fungal pathogenesis, we have identified the gene encoding a RAS homologue in this organism. The RAS1 gene was disrupted by transformation and homologous recombination. The resulting ras1 mutant strain was viable, but failed to grow at 37 degrees C, and exhibited significant defects in mating and agar adherence. The ras1 mutant strain was also avirulent in an animal model of cryptococcal meningitis. Reintroduction of the wild-type RAS1 gene complemented these ras1 mutant phenotypes and restored virulence in animals. A dominantly active RAS1 mutant allele, RAS1Q67L, induced a differentiation phenotype known as haploid fruiting, which involves filamentation, agar invasion and sporulation in response to nitrogen deprivation. The ras1 mutant mating defect was suppressed by overexpression of MAP kinase signalling elements and partially suppressed by exogenous cAMP. Additionally, cAMP also suppressed the agar adherence defect of the ras1 mutant. However, the ability of the ras1 mutant strain to grow at elevated temperature was not restored by cAMP or MAP kinase overexpression. Our findings support a model in which RAS1 signals in C. neoformans through cAMP-dependent, MAP kinase, and RAS-specific signalling cascades to regulate mating and filamentation, as well as growth at high temperature which is necessary for maintenance of infection.

Animals↗
PubMed2000

Characterization of the MFalpha pheromone of the human fungal pathogen cryptococcus neoformans.

Cryptococcus neoformans is an important human pathogenic fungus with a defined sexual cycle and well-developed molecular and genetic approaches. C. neoformans is predominantly haploid and has two mating types, MATa and MATalpha. Mating is known to be regulated by nutritional limitation and thought also to be regulated by pheromones. Previously, a portion of the MATalpha locus was cloned, and a presumptive pheromone gene, MFalpha1, was identified by its ability to induce conjugation tube-like filaments when introduced by transformation into MATa cells. Here, the ability of the MFalpha1 gene to induce these morphological changes in MATa cells was used as a phenotypic assay to perform a structure-function analysis of the gene. We show that the MFalpha1 open reading frame is required for the morphological response of MATa cells. We also find that the cysteine residue of the C-terminal CAAX motif is required for activity of the MFalpha1 pheromone. In addition, we use a reporter system to measure the expression levels of the MFalpha1 pheromone gene and find that two signals, nutrient starvation and the presence of factors secreted by mating partner cells, impinge on this promoter and regulate MFalpha1 expression. We identify a second pheromone gene, MFalpha2, and show phenotypically that this gene is also expressed. Finally, we have synthesized the MFalpha1 pheromone and show that only the predicted mature modified form of the alpha-factor peptide triggers morphological responses in MATa cells.

Cryptococcus neoformans↗